PROJECT SUMMARY Our goal is to develop a mechanistic understanding of the inhibition of synaptic vesicle fusion by monomeric -Synuclein (S). This research will establish the foundation for new therapeutic strategies in the treatment of Parkinson's disease (PD). There is growing consensus that monomeric S is a central regulatory component of synaptic vesicle trafficking. Although the formation of Lewy bodies, mediated by the aggregation of S into insoluble fibrils, is commonly associated with PD, high levels of S have also been shown to disrupt normal vesicle trafficking and markedly inhibit neurotransmitter release without the formation of S aggregates. Our approach will involve quantitative studies of the biophysical and mechanical properties of synaptic vesicle membranes for which we will combine coarse-grained molecular dynamics simulations with a panel of complementary biophysical experiments. A precise understanding of the native interactions between monomeric S and synaptic vesicle membranes will position us to evaluate the protein's role in vesicle trafficking defects as they relate to PD. Limited biophysical data have yielded conflicting views on how S over-expression inhibits vesicle trafficking and fusion in the absence of fibril formation. In multiple model systems from yeast to rodents, an overabundance of S has been shown to stall proper synaptic vesicle cycling at the plasma membrane. One view is that this pathology may be driven by interactions between S and other synaptic or plasma membrane proteins (e.g. SNARES). We propose an alternate view based on recent work both from our labs and others. According to this view, S can directly alter the physical properties of lipids within membranes - namely membrane rigidity and phase. This is achieved in the absence of specific interactions with other proteins. We therefore reason that S might have an intrinsic capacity to control synaptic vesicle fusion. This hypothesis is motivated by our preliminary data and published result from biophysical experiments, which show that S reduces a membrane's rigidity, can alter membrane curvature, and can inhibit fusion of synthetic (otherwise protein-free) lipid vesicles. Our proposed research intimately combines computational modeling with experimental x-ray scattering and atomic force microscopy to bridge a critical gap in understanding how S creates physical barriers to vesicle fusion. The proposed research avenue will provide critical information about PD associated trafficking defects. Ultimately, our work will lead to better understanding of normal and abnormal functions of S and position the community to develop new therapeutic strategies that exploit the native state of the protein (i.e., restoring proper vesicle trafficking).